Combination therapy with a compound acting as a platelet adp receptor inhibitor

ABSTRACT

The present invention is directed to pharmaceutical compositions and methods of using combination therapies containing [4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea, or a pharmaceutically acceptable salt thereof, for the treatment of thrombosis diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Nos. 60/915,649, filed on May 2, 2007,60/915,911, filed on May 3, 2007, 60/947,921, filed on Jul. 3, 2007, and60/978,700, filed on Oct. 9, 2007, all of which are hereby incorporatedby reference in their entirety.

FIELD OF INVENTION

The present invention relates generally to novel compositions andmethods of using a combination of a platelet ADP receptor inhibitor,[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea(Compound A), and an anticoagulant agent or another antiplatelet agentfor the treatment of thrombotic diseases. The present invention alsorelates to novel compositions and methods using a combination ofCompound A with an anticoagulant and another antiplatelet agent for thetreatment of thrombotic diseases.

BACKGROUND OF THE INVENTION

Thrombotic complications are a major cause of death in theindustrialized world. Examples of these complications include acutemyocardial infarction, unstable angina, chronic stable angina, transientischemic attacks, strokes, peripheral vascular disease,preeclampsia/eclampsia, deep venous thrombosis, embolism, disseminatedintravascular coagulation and thrombotic cytopenic purpura. Thromboticand restenotic complications also occur following invasive procedures,e.g., angioplasty, carotid endarterectomy, post CABG (coronary arterybypass graft) surgery, vascular graft surgery, stent placements andinsertion of endovascular devices and prostheses. It is generallythought that platelet aggregates play a critical role in these events.Blood platelets, which normally circulate freely in the vasculature,become activated and aggregate to form a thrombus with disturbed bloodflow caused by ruptured atherosclerotic lesions or by invasivetreatments such as angioplasty, resulting in vascular occlusion.

An important mediator of platelet activation and aggregation is ADP(adenosine 5′-diphosphate) which is released from blood platelets in thevasculature upon activation by various agents, such as collagen andthrombin, and from damaged blood cells, endothelium or tissues.Activation of ADP results in the recruitment of more platelets andstabilization of existing platelet aggregates. Adenosine nucleotidesthat are released following platelet activation signal through the P2purinergic receptors on the platelet membrane (Mills, D. C. Thromb.Haemost. 1996, 76:835-56; Gachet, C. Annu Rev Pharmacol Toxicol 2006,46:277-300). P2 receptors are classified as either ligand-gated ionchannels (P2X) or G-protein coupled receptors (GPCRs) designated as P2Yreceptors (Abbrachio, M. P., Burnstock, G. Pharmacol Ther 1994,64:445-75). Although initially thought to mediate its effects through asingle receptor (termed P2Y_(ADP) (Fredholm, B. B. et al, TIPS 1997,18:79-82), ADP has more recently been shown to act on platelets throughtwo GPCRs, the G_(q)-coupled P2Y₁ receptor, and the G_(i)-coupled P2Y₁₂receptor. The P2Y₁₂ receptor was identified through expression cloning(Hollopter, G. et al, Nature 2001, 409:202-07), and has beendemonstrated to play a critical role in thrombus stability (Andre, P. etal, J Clin Inves, 2003, 112:398-406) and is the target of thethienopyridine drugs ticlopine and clopidogrel. ATP, on the other hand,acts through the ligand-gated channel P2X1 on platelets (Gachet, C. AnnuRev Pharmacol Toxicol 2006, 46:277-300).

U.S. Patent Publication US 2007/0123547, titled“[4-(6-Halo-7-substituted-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureasAnd Forms And Methods Related Thereto,” filed Nov. 3, 2006, the contentsof which are incorporated herein by reference in its entirety, disclosesa platelet ADP receptor inhibitor compound,[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,(Compound A), which has the following structure:

and acts as a specific antagonist of P2Y₁₂.

Since treatment for diseases such as acute coronary syndrome mightrequire coadministration of an antiplatelet agent and an anticoagulantagent, a combination would allow for increased efficacy and may providean improved safety profile. Thus, there is a need for combinationtherapies combining an antiplatelet agent with an anticoagulant agentthat have enhanced efficacy. There is also a need for a combinationtherapy that allows for lower (i.e. sub-therapeutic) dosages of eachindividual agent to be used in the combination which may provide animproved safety profile.

There is also a need for combination of two different antiplatelet drugsthat act by different mechanisms (e.g., a P2Y₁₂ antagonist (Compound A)and a cox-1 inhibitor (aspirin)) in combination with an anticoagulant,as such a triple combination (clopidogrel, aspirin and heparin) ispresently used in the clinic (as separate entities) during angioplastyprocedures and has been found to be more efficatious than any of thesedrugs used alone or a combination of any two of these agents.

SUMMARY OF THE INVENTION

This invention provides methods and pharmaceutical compositions ofcombined therapies comprising a P2Y₁₂ antagonist, having the structure:

which has the chemical name[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,and is referred to throughout as “Compound A”.

It is contemplated based on experimental results that a combination ofCompound A with an anticoagulant agent, such as a factor Xa inhibitor,and/or another antiplatelet agent, such as a cyclooxygenase inhibitor,will produce improved antithrombotic effect over any of the agentsalone.

Accordingly, the present invention provides novel methods for treating acondition in a mammal characterized by undesired thrombosis, comprisingadministering to said mammal a therapeutically effective amount ofCompound A,[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of another therapeutic agent. The other therapeuticallyeffective agent is selected from an anticoagulant agent, an antiplateletagent, or combinations thereof.

In one aspect, the present invention provides a novel method forpreventing or treating thrombosis and thrombosis-related conditions in amammal comprising administering to said mammal a therapeuticallyeffective amount of[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea(Compound A), or a pharmaceutically acceptable salt thereof, and ananticoagulant agent. In some embodiments, the anticoagulant agent is aspecific inhibitor of factor Xa,[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide(betrixaban, see below), or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a novel method forpreventing or treating thrombosis and/or a condition in a mammalcomprising administering to said mammal a therapeutically effectiveamount of[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea(Compound A), or a pharmaceutically acceptable salt thereof, and anotherantiplatelet agent.

In still another aspect, the present invention provides a novel methodfor preventing or treating thrombosis and/or a thrombosis-relatedcondition in a mammal comprising administering to said mammal atherapeutically effective amount of Compound A or a pharmaceuticallyacceptable salt thereof, an anticoagulant agent and another antiplateletagent.

The present invention also provides a novel pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, Compound A,[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of another therapeutic agent. The other therapeuticallyeffective agent is selected from an anticoagulant agent, an antiplateletagent, or combinations thereof.

In another aspect, the present invention provides a novel pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, Compound Aor a pharmaceutically acceptable salt thereof, and an anticoagulantagent. In some embodiments, the anticoagulant agent is betrixaban, or apharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a novel pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, Compound Aor a pharmaceutically acceptable salt thereof, and another antiplateletagent.

In still another aspect, the present invention provides a novelcomposition comprising a pharmaceutically acceptable carrier, Compound Aor a pharmaceutically acceptable salt thereof, an anticoagulant agentand another antiplatelet agent.

This invention also provides a novel kit, comprising: a first containerfor containing Compound A, or a pharmaceutically acceptable saltthereof, and a second container for containing another therapeutic agentselected from the group consisting of an anticoagulant agent, anantiplatelet agent other than Compound A, and combinations thereof.

These and other embodiments of the present invention are furtherdescribed in the text that follows.

The compositions of this invention are contemplated to provide for asynergistic effect in one or more of the following areas: improvedtherapeutic results, improved safety, reduced amount to achieveequivalent efficacy of one or more of the combination drugs as comparedto the amount of that drug required to achieve the same level ofefficacy when used alone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the percent inhibition of thrombus formation by acombination of 1.1 μM of Compound A and a coagulation factor Xainhibitor, betrixaban, in a perfusion chamber assay, where theconcentration of betrixaban varies from 100 nM to 1.1 μM.

FIG. 2 shows the dose responsive inhibition of thrombosis by acombination of a fixed concentration of Compound A with increasingconcentrations of betrixaban upon perfusion of whole human blood over acollagen coated surface where the concentration of betrixaban variesfrom 3 nM to 1.1 μM.

FIG. 3 shows dose responsive inhibition of thrombosis by a combinationof increasing concentrations of Compound A with a fixed concentration ofbetrixaban upon perfusion of whole human blood over collagen coatedsurface.

FIG. 4 shows combined inhibition of platelet mediated thrombingeneration by the combination of inhibition of the platelet P2Y₁₂receptor by Compound A and coagulation by a factor Xa inhibitor,betrixaban.

FIG. 5 shows the combined inhibitory effect of a coagulation factor XIinhibitor (an anti-factor XI antibody) and Compound A on plateletthrombus formation under conditions where either single agent alone didnot show inhibition.

FIG. 6 shows the combined inhibitory effect of Compound A and acoagulation factor XI inhibitor (anti-factor XI antibody) on plateletthrombus formation on a collagen: tissue factor surface, whereasCompound A alone or a combination of Compound A and betrixaban did notshow inhibition.

FIG. 7 shows the combined inhibitory effect of increasing concentrationsof Compound A with a direct thrombin inhibitor bivalirudin (12 μg/mL) onthrombus formation under arterial shear conditions.

FIG. 8 shows the combined effect of Compound A and aspirin, in thepresence of a factor Xa inhibitor (5 μM of C921-78 (see Betz A., Wong P.W., Sinha U. Inhibition of factor Xa by a peptidyl-alpha-ketothiazoleinvolves 2 steps: evidence for a stabilizing conformational change.Biochemistry 1999; 38: 14582-14591, incorporated herein by reference inits entirety)) on inhibition of the thrombotic process in a whole bloodperfusion chamber assay.

FIG. 9 shows combined effect of P2Y₁₂ inhibition by Compound A andinhibition of TP receptors by ifetroban in the presence of a factor Xainhibitor (5 μM of C921-78) on inhibition of the thrombotic process in awhole blood perfusion chamber assay.

FIGS. 10-13 shows the effect of Compound A in an intravital microscopymodel.

FIG. 10 shows that Compound A delays the time for appearance of firstthrombus in the intravital microscopy model. FIG. 11 shows thepharmacokinetics and pharmacodynamics (PK/PD) correlation of Compound Ain delaying the time for appearance of first thrombus.

FIG. 12 shows that Compound A inhibits vascular occlusion. FIG. 13 showsthe PK/PD correlation of Compound A in inhibiting vascular occlusion.

FIGS. 14-17 shows the effect of a combination of Compound A andbetrixaban in the same intravital microscopy model. FIGS. 14 and 15 showthat the combination of non-effective doses of Compound A and betrixabansignificantly prolongs time for appearance of first thrombus. FIGS. 16and 17 show that the combination of non-effective doses of Compound Aand betrixaban significantly inhibits of thrombosis.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a method and compositions for preventing ortreating thrombosis and thrombosis-related conditions in a mammal usinga combination of Compound A with a co-administered agent. Prior todescribing this invention in more detail, the following terms aredefined.

I. DEFINITIONS

It is to be noted that as used herein and in the claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “apharmaceutically acceptable carrier” in a composition includes two ormore pharmaceutically acceptable carriers, and so forth.

It must be further noted that the classification of certain therapeuticagents based on their intended use or mechanisms of action is based onthe general knowledge of a person skilled in the art and forclassification purposes only. The purported mechanisms are not intendedto be used as a limitation for the therapeutic agents unless the contextclearly dictates otherwise. Some therapeutic agents may act through twoor more mechanisms or are able to be used to treat two or moreconditions. It is also to be understood that the particular agents givenin each categories are for examples only and are not intended to limitthe scope of the present invention.

“Comprising” is intended to mean that the compositions and methodsinclude the recited elements, but do not exclude others. “Consistingessentially of” when used to define compositions and methods, shall meanexcluding other elements of any essential significance to thecombination for the intended use. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

The term “treatment” or “treating” means any treatment of a disease orcondition in a subject, such as a mammal, including: 1) preventing orprotecting against the disease or condition, that is, causing theclinical symptoms not to develop; 2) inhibiting the disease orcondition, that is, arresting or suppressing the development of clinicalsymptoms; and/or 3) relieving the disease or condition that is, causingthe regression of clinical symptoms.

As used herein, the term “preventing” refers to the prophylactictreatment of a patient in need thereof. The prophylactic treatment canbe accomplished by providing an appropriate dose of a therapeutic agentto a subject at risk of suffering from an ailment, thereby substantiallyaverting onset of the ailment.

It will be understood by those skilled in the art that in humanmedicine, it is not always possible to distinguish between “preventing”and “suppressing” since the ultimate inductive event or events may beunknown, latent, or the patient is not ascertained until well after theoccurrence of the event or events. Therefore, as used herein the term“prophylaxis” is intended as an element of “treatment” to encompass both“preventing” and “suppressing” as defined herein. The term “protection,”as used herein, is meant to include “prophylaxis.”

The term “mammal” includes, without limitation, human, monkeys, rabbits,mice domestic animals, such as dogs and cats, farm animals, such ascows, horses, or pigs, and laboratory animals.

The term “condition” refers to a disease state for which the methods andcompositions of the present invention are being used against.

As used herein, “thrombosis and thrombosis-related conditions” may beany of but are not limited to the following: any thrombosis,particularly a platelet-dependent thrombotic indication, including, butnot limited to, acute myocardial infarction, unstable angina, chronicstable angina, transient ischemic attacks, strokes, peripheral vasculardisease, preeclampsia/eclampsia, deep venous thrombosis, embolism,disseminated intravascular coagulation and thrombotic cytopenic purpura,thrombotic and restenotic complications following invasive procedures,e.g., angioplasty, carotid endarterectomy, post CABG (coronary arterybypass graft) surgery, vascular graft surgery, stent placements andinsertion of endovascular devices and prostheses, and hypercoagulablestates related to genetic predisposition or cancers.

“[4-(6-Fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,”or “Compound A” is intended to refer to the compound having thefollowing structure:

and its tautomers.

“Therapeutically effective amount” means an amount of Compound A or theco-administered agent of the present invention that is effective totreat a target disease or condition when administered in combination.The therapeutically effective amount will vary depending upon thespecific combination, the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the dosing regimen to be followed, timing of administration,the manner of administration and the like, all of which can bedetermined readily by one of ordinary skill in the art.

In some embodiments, it is contemplated that the therapeuticallyeffective amount of Compound A or the co-administered agent in thecombination can be less than their respective effective amount when usedas a single agent. In this case, the therapeutically effective amount isreferred to as “sub-therapeutic dosage.” Thus, the term “sub-therapeuticdosage” is intended to mean a dosage that is lower than the optimaldosage for a therapeutic agent when used as a single agent, but whenused in the combinations described herein, provides a therapeuticresult.

“Anticoagulant agents” or “anticoagulants” are agents that prevent bloodclot formation. Examples of anticoagulant agents include, but are notlimited to, specific inhibitors of thrombin, factor IXa, factor Xa,factor XI, factor XIa, factor XIIa or factor VIa, heparin andderivatives, vitamin K antagonists, and anti-tissue factor antibodies,as well as inhibitors of P-selectin and PSGL-1. Examples of specificinhibitors of thrombin include hirudin, bivalirudin (Angiomax®),argatroban, ximelagatran (Exanta®, see structure below), dabigatran (seestructure below), AZD0837 (being studied in clinical trial A Controlled,Randomized, Parallel, Multi-Centre Feasibility Study of the Oral DirectThrombin Inhibitor, AZD0837, Given as ER Formulation, in the Preventionof Stroke and Systolic Embolic Events in Patients With AtrialFibrillation, Who Are Appropriate for But Unable/Unwilling to Take VKATherapy with ClinicalTrials.gov Identifier: NCT00623779), RB2006 (asingle-stranded, nucleic acid aptamer, by Regado Biosciences, Durham,N.C., as described in Dyke, C. K. et al., First-in-Human Experience ofan Antidote-Controlled Anticoagulant Using RNA Aptamer Technology,Circulation 2006; 114:2490-2497), and lepirudin (Refludan®). Examples ofheparin and derivatives include unfractionated heparin (UFH), lowmolecular weight heparin (LMWH), such as enoxaparin (Lovenox®),dalteparin (Fragmin®), and danaparoid (Orgaran®); and syntheticpentasaccharide, such as fondaparinux (Arixtra®), idraparinux andbiotinylated idraparinux. Examples of vitamin K antagonists includewarfarin (Coumadin®), phenocoumarol, acenocoumarol (Sintrom®),clorindione, dicumarol, diphenadione, ethyl biscoumacetate,phenprocoumon, phenindione, and tioclomarol.

The term “factor Xa inhibitors” or “inhibitors of factor Xa” refers tocompounds that can inhibit the coagulation factor Xa's activity ofcatalyzing conversion of prothrombin to thrombin in vitro and/or invivo. Factor Xa is an enzyme in the coagulation pathway, and is theactive component in the prothrombinase complex that catalyzes theconversion of prothrombin to thromin. Thrombin is responsible forconverting fibrinogen to fibrin, and leads to formation of blood clot.Thus, inhibition of factor Xa is considered to be an effective strategyof treating and preventing thrombotic disease(s). A preferred factor Xainhibitor inhibits thrombin formation both in vitro and in vivo. A morepreferred factor Xa inhibitor shows anticoagulant efficacy in vivo. Theterm “specific inhibitor of factor Xa” or “specific factor Xa inhibitor”is intended to refer to factor Xa inhibitors that exhibit substantiallyhigher inhibitory activities against factor Xa than against otherenzymes or receptors of the same mammal. Preferably, a specific factorXa inhibitor does not have significant known inhibitory activity againstother enzymes or receptors in the same mammal system at itstherapeutically effective concentrations.

Examples of known factor Xa inhibitors include, without limitation,fondaparinux, idraparinux, biotinylated idraparinux, enoxaparin,fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor, LY517717 (byEli Lilly & Co., Indianapolis, Ind., USA, having the structure ofN-{(1R)-2-[4-(1-methyl-4-piperidinyl)-1-piperazinyl]-2-oxo-1-phenylethyl}-1H-indole-6-carboxamide,as described in, e.g., A Phase II Study of the Oral Factor Xa InhibitorLY517717 for the Prevention of Venous Thromboembolism after Hip or KneeReplacement, Agnelli G. et al, J. Thromb. Haemost. 2007, 5(4):746-53,studied in clinical trials, such as A Comparison of the OralAnticoagulant LY517717 Difumarate to Subcutaneous Enoxaparin for thePrevention of Venous Thromboembolic Events (VTE) Post-Total HipReplacement (THR) and Post-Total Knee Replacement (TKR) Surgery, withClinicalTrials.gov Identifier: NCT00074828), YM-150 (as described ine.g., Eriksson, B. I. et al, J. Thromb. Haemost. 2007, 5:1660-65, andstudied in clinical trials, such as Direct Factor Xa Inhibitor YM150 forPrevention of Venous Thromboembolism in Patients Undergoing ElectiveTotal Hip Replacement. A Double Blind, Parallel, Dose-Finding Study inComparison With Open Label Enoxaparin with ClinicalTrials.govIdentifier: NCT00353678), Daiichi DU-176b (as described in, e.g., E.Hylek, DU-176b, An Oral, Direct Factor Xa Antagonist, Current Opinion inInvestigational Drugs 2007 8:778-783 and studied in clinical trials,such as, A Phase IIb, Randomized, Parallel Group, Double-Blind,Double-Dummy, Multi-Center, Multi-National, Multi-Dose, Study of DU-176bCompared to Dalteparin in Patients Undergoing Elective Unilateral TotalHip Replacement with ClinicalTrials.gov Identifier: NCT00398216),betrixaban (as described below), and compounds listed in Table 1, andderivatives thereof.

TABLE 1 Structure Chemical Name

(5S)-5-chloro-N-((2-oxo-3-(4-(3-oxomorpholino)phenyl)-oxazolidin-5-yl)methyl)thiophene-2-carboxamideRivaroxaban,as described in e.g.,Turpie, A. G., et al, J.Thromb.Haemost.2005, 3(11): 2479-86

1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-3a,4,5,6,7,7a-hexahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamideApixaban

1-(3-aminobenzo[d]isoxazol-5-yl)-N-(4-(2-((dimethylamino)methyl)-1H-imidazol-1-yl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamideRazaxaban

(E)-2-(5-chlorothiophen-2-yl)-N-((S)-1-((S)-1-morpholino-1-oxopropan-2-yl)-2-oxopyrrolidin-3-yl)ethenesulfonamide

(R)-N-(2-(4-(1-methylpiperidin-4-yl)piperazin-1-yl)-2-oxo-1-phenylethyl)-1H-indole-6-carboxamideas described in e.g.,Agnelli, G. et al, J.Thromb. Haemost.2007 5(4):746-53

(2R,4R)-N1-(4-chlorophenyl)-N2-(2-fluoro-4-(2-oxopyridin-1(2H)-yl)phenyl)-4-methoxypyrrolidine-1,2-dicarboxamideas described in e.g.,Pipeline Insight:Antithrombotics -ReachingtheUntreated ProphylaxisMarket, 2007

(S)-3-(7-carbamimidoylnaphthalen-2-yl)-2-(4-((S)-1-(1-iminoethyl)pyrrolidin-3-yloxy)phenyl)propanoicacidas described in e.g.,Herbert, J. M., et al, JPharmacol Exp Ther.1996276(3): 1030-8

2-(N-((7-carbamimidoylnaphthalen-2-yl)methyl)-N-(4-(1-(1-iminoethyl)piperidin-4-yloxy)phenyl)sulfamoyl)-aceticacid as described in e.g.,Taniuchi, Y., et al,Thromb Haemost.1998 79(3):543-8

methyl (2R,3R)-2-(3-carbamimidoylbenzyl)-3-[[4-(1-oxidopyridin-4-yl)benzoyl]amino]butanoateOtamixaban

The term“[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide,”is intended to refer to the compound having the following structure ortautomers thereof, which is also referred to herein as betrixaban:

Betrixaban is described in U.S. Patent Application PublicationUS2007/0112039, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/735,224 filed Nov. 8, 2005, the contents of which areincorporated herein by reference in their entirety. Betrixaban is knownto be a specific inhibitor of factor Xa.

The term “factor XI inhibitors” or “inhibitors of factor XI” arecompounds that can inhibit the coagulation factor XI. Upon proteolyticactivation, factor XI is converted to the active enzyme factor XIa,which cleaves factor IX into factor IXa. Factor IXa then hydrolyzesfactor X to factor Xa, which initiates the coagulation reactions thatleads to blood clot formation as described above. An anti-factor XIantibody is a protein produced by an immune response that specificallybinds factor XI, thus inhibits its activity. Some anti-factor XIantibodies are available commercially from, such as HaematologicTechnologies, Essex Junction, Vt., USA.

“Injectable anticoagulants” are anticoagulant agents that areadministrated to a mammal through injections. Examples of injectableanticoagulants are unfractionated heparin, low molecular weightheparins, and synthetic pentasaccarides.

“Antiplatelet agents” or “platelet inhibitors” are agents that block theformation of blood clots by preventing the aggregation of platelets.There are several classes of antiplatelet agents based on theiractivities, including, GP IIb/IIIa antagonists, such as abciximab(ReoPro®), eptifibatide (Integrilin®), and tirofiban (Aggrastat®); P2Y₁₂receptor antagonists, such as clopidogrel (Plavix®), ticlopidine(Ticlid®), cangrelor, ticagrelor, and prasugrel; phosphodiesterase III(PDE III) inhibitors, such as cilostazol (Pletal®), dipyridamole(Persantine®) and Aggrenox® (aspirin/extended-release dipyridamole);thromboxane synthase inhibitors, such as furegrelate, ozagrel, ridogreland isbogrel; thromboxane A2 receptor antagonists (TP antagonist), suchas ifetroban, ramatroban, terbogrel,(3-{6-[(4-chlorophenylsulfonyl)amino]-2-methyl-5,6,7,8-tetrahydronaphth-1-yl}propionicacid (also known as Servier S 18886, by de Recherches InternationalesServier, Courbevoie, France); thrombin receptor antagonists, such asSCH₅₃₀₃₄₈ (having the chemical name of ethyl(1R,3aR,4aR,6R,8aR,9S,9aS)-9-((E)-2-(5-(3-fluorophenyl)pyridin-2-yl)vinyl)-1-methyl-3-oxododecahydronaphtho[2,3-C]furan-6-ylcarbamate, by Schering Plough Corp., New Jersey, USA,described in US20040192753A1 and US2004/0176418A1 and studied inclinical trials, such as A Multicenter, Randomized, Double-Blind,Placebo-Controlled Study to Evaluate the Safety of SCH 530348 inSubjects Undergoing Non-Emergent Percutaneous Coronary Intervention withClinicalTrials.gov Identifier: NCT00132912); P-selectin inhibitors, suchas2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[H]quinoline-4-carboxylicacid (also known as PSI-697, by Wyeth, New Jersey, USA); andnon-steroidal anti-inflammatory drugs (NSAIDS), such as acetylsalicylicacid (Aspirin®), resveratrol, ibuprofen (Advil®, Motrin®), naproxen(Aleve®, Naprosyn®), sulindac (Clinoril®), indomethacin (Indocin®),mefenamate, droxicam, diclofenac (Cataflam®, Voltaren®), sulfinpyrazone(Anturane®), and piroxicam (Feldene®). Among the NSAIDS, acetylsalicylicacid (ASA), resveratrol and piroxicam are preferred. Some NSAIDS inhibitboth cyclooxygenase-1 (cox-1) and cyclooxygenase-2 (cox-2), such asaspirin and ibuprofen. Some selectively inhibit cox-1, such asresveratrol, which is a reversible cox-1 inhibitor that only weaklyinhibits cox-2. Beta blockers and calcium channel blockers, which aredescribed below, also have a platelet-inhibiting effect.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on theparticular therapeutic agents described herein. When therapeutic agentsdescribed in the invention contain relatively acidic functionalities,base addition salts can be obtained by contacting the neutral form ofsuch compounds with a sufficient amount of the desired base, either neator in a suitable inert solvent. Examples of salts derived frompharmaceutically-acceptable inorganic bases include aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, zinc and the like. Salts derived frompharmaceutically-acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. When therapeutic agentsdescribed in the invention contain relatively basic functionalities,acid addition salts can be obtained by contacting the neutral form ofsuch compounds with a sufficient amount of the desired acid, either neator in a suitable inert solvent. Examples of pharmaceutically acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, malonic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, e.g., Berge, S.M., et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science,1977, 66: 1-19). Certain specific therapeutic agents contain both basicand acidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

Certain preferred salt forms for Compound A are described in U.S. PatentApplication Publication US 2007/0123547, titled“[4-(6-Halo-7-substituted-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureasAnd Forms And Methods Related Thereto,” and filed Nov. 3, 2006, andclaims priority from Provisional Application 60/733,650, filed on Nov.3, 2005, both of which are hereby incorporated by reference in theirentirety. Preferably, Compound A forms a potassium salt (Formula I):

or a sodium salt (Formula II):

Several crystalline solid or amorphous forms of the potassium saltFormula I and sodium salt Formula II are also described in U.S. PatentApplication Publication US 2007/0123547. Some preferred crystallinesolid forms of the potassium salt Formula I have at least one of thefollowing characteristics: (1) an infrared spectrum comprising peaks atabout 3389 cm⁻¹ and about 1698 cm⁻¹; (2) an X-ray powder diffractionpattern comprising peaks at about 9.5 and about 25.5 ° 2θ; and (3) a DSCmaximum endotherm at about 246° C. Among these forms, some have an infrared spectrum comprising absorption peaks at about 3559, 3389, 3324,1698, 1623, 1563, 1510, 1448, 1431, 1403, 1383, 1308, 1269, 1206, 1174,1123, 1091, 1072, 1030, 987, 939, 909, 871, 842, 787, 780, 769, 747,718, 701, 690 and 667 cm⁻¹. Other preferred crystalline solid forms ofthe potassium salt Formula I have at least one of the followingcharacteristics: (1) an infrared spectrum comprising peaks at about 3327cm⁻¹ and about 1630 cm⁻¹; (2) an X-ray powder diffraction patterncomprising peaks at about 20.3 and about 25.1 ° 2θ; and (3) a DSCmaximum endotherm at about 293° C. Among these forms, some have an infrared spectrum comprising absorption peaks at about 3584, 3327, 3189,2935, 2257, 2067, 1979, 1903, 1703, 1654, 1630, 1590, 1557, 1512, 1444,1429, 1406, 1375, 1317, 1346, 1317, 1288, 1276, 1243, 1217, 1182, 1133,1182, 1133, 1093, 1072, 1033, 987, 943, 907, 883, 845, 831, 805, 776,727, 694 and 674 cm⁻¹. Some preferred amorphous forms of the sodium saltFormula II have at least one of the following characteristics: (1) aninfrared spectrum comprising peaks at about 3360, 1711, 1632, 1512,1227, 1133 and 770 cm⁻¹; and (2) an X-ray powder diffraction patterncomprising a broad peak substantially between about 15 and about 30 °2θ. Among these forms, some have an infra red spectrum comprisingabsorption peaks at about 3360, 1711, 1632, 1556, 1512, 1445, 1407,1375, 1309, 1280, 1227, 1133, 1092, 1032, 987, 905, 781, 770 and 691cm⁻¹.

Certain preferred salt forms for betrixaban are disclosed in U.S. PatentApplication Publication US2007/0112039. In particular, the applicationdiscloses that betrixaban forms a salt with an acid. The acid ispreferably selected from the group consisting of hydrochloric, lactic,maleic, phenoxyacetic, propionic, succinic, adipic, ascorbic, camphoric,gluconic, phosphic, tartaric, citric, methanesulfonic, fumaric,glycolic, naphthalene-1,5-disulfonic, gentisic and benzenesulfonic.Preferably the acid is selected from the group consisting ofhydrochloric, lactic, maleic, phenoxyacetic, propionic, and succinic.Most preferably, the acid is maleic acid, forming the maleate salt ofbetrixaban. One embodiment of the maleate salt of betrixaban exists asFormula III

Further the salt of Formula III may exist in a crystalline polymorph asdisclosed in U.S. Patent Application Publication US2007/0112039. Onecrystalline polymorph form of Formula III exhibits a powder X-raydiffraction pattern having at least four and preferably eight of thefollowing approximate characteristic peak locations: 4.9, 9.7, 13.8,14.1, 15.2, 17.6, 18.5, 20.8, 21.6, 22.7, 24.1, 26.3, 26.8 degrees 2θ.In a more preferred crystalline polymorph form, the powder X-raydiffraction pattern has approximate characteristic peak locations of4.9, 9.7, 11.8, 13.8, 14.1, 15.2, 17.6, 18.5, 19.9, 20.8, 21.6, 22.7,24.1, 25.0, 26.3, 26.8 degrees 2θ.

The neutral forms of the therapeutic agents may be regenerated bycontacting the salt with a base or acid and isolating the parenttherapeutic agent in the conventional manner. The parent form of thetherapeutic agent differs from the various salt forms in certainphysical properties, such as solubility in polar solvents, but otherwisethe salts are equivalent to the parent form for the purposes of thepresent invention.

In addition to salt forms, certain therapeutic agents are in a prodrugform. Prodrugs of the therapeutic agents are those compounds thatreadily undergo chemical changes under physiological conditions toprovide the compound having therapeutic activities. Additionally,prodrugs can be converted to the active compound by chemical orbiochemical methods in an ex vivo environment. For example, prodrugs canbe slowly converted to the active compound described in the inventionwhen placed in a transdermal patch reservoir with a suitable enzyme orchemical reagent.

Certain therapeutic agents described in the invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms are equivalent to unsolvated forms and areintended to be encompassed within the scope of the present invention.Certain therapeutic agents may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for theuses contemplated by the present invention and are intended to be withinthe scope of the present invention.

“Pharmaceutically acceptable carriers” refer to any diluents,excipients, or carriers that may be used in the compositions of theinvention. Pharmaceutically acceptable carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances, such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. They are preferably selected with respect to theintended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

II. DETAILED DESCRIPTION OF THE EMBODIMENTS

a. Methods of Treatment

The present invention provides novel methods for treating a condition ina mammal characterized by undesired thrombosis comprising administeringto said mammal a therapeutically effective amount of the followingtherapeutic agents:

(1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof, and

(2) a second therapeutic agent selected from the group consisting of ananticoagulant, an antiplatelet agent, or combinations thereof.

In one aspect, the invention provides a novel method for preventing ortreating thrombosis and/or a thrombosis-related condition in a mammalcomprising administering to said mammal a therapeutically effectiveamount of the following two therapeutic agents:

(1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof, and

(2) an anticoagulant agent.

In some embodiments, the anticoagulant agent is an inhibitor of factorXa. In some embodiments, the factor Xa inhibitor is a specific factor Xainhibitor.

In some embodiments, the factor Xa inhibitor is YM-150, Daiichi DU-176b,LY517717, or a compound selected from Table 1.

In some embodiments, the factor Xa inhibitor is rivaroxaban.

In some embodiments, the specific inhibitor of factor Xa is betrixaban,or a pharmaceutically acceptable salt thereof. In still someembodiments, the pharmaceutically acceptable salt of betrixaban is themaleate salt.

In other embodiments, the anticoagulant agent is selected from the groupconsisting of specific inhibitors of thrombin, factor IXa, factor XI,factor XIa or factor VIIa. In some embodiments, the anticoagulant agentis selected from the group consisting of bivalirudin, argatroban,lepirudin, warfarin, ximelagatran, AZD0837, RB2006, dabigatran andphenocoumarol.

In still other embodiments, the anticoagulant agent is an injectableanticoagulant agent. In some embodiments, the anticoagulant agent isselected from the group consisting of synthetic pentasaccharides and lowmolecular weight heparin. In some embodiments, the anticoagulant agentis selected from the group consisting of fondaparinux, danaparoid,enoxaparin, dalteparin and unfractionated heparin.

In other embodiments, the anticoagulant agent is an anti-factor XIantibody. In still other embodiments, the anticoagulant agent isbivalirudin.

In another aspect, the invention provides a method for treating acondition in a mammal characterized by undesired thrombosis in a mammalcomprising administering to said mammal a therapeutically effectiveamount of the following two therapeutic agents:

-   -   (1)        [4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,        or a pharmaceutically acceptable salt thereof, and    -   (2) another antiplatelet agent.

In some embodiments, the antiplatelet agent is an antagonist of the TPreceptor. In some embodiments, the antagonist of the TP receptor isifetroban. In other embodiments, the antiplatelet agent is acyclooxygenase inhibitor. In some embodiments, the cyclooxygenaseinhibitor is acetylsalicylic acid. In some embodiments, the antiplateletagent is a reversible cyclooxygenase-1 inhibitor. In some embodiments,the reversible cyclooxygenase-1 inhibitor is resveratrol.

In some embodiments, the other antiplatelet agent is selected from thegroup consisting of abciximab, eptifibatide, tirofiban, dipyridamole,aggrenox, cilostazol, isbogrel, furegrelate and ozagrel.

In some embodiments, at least one of the therapeutic agents isadministered in a sub-therapeutic dosage.

In some embodiments, both of the therapeutic agents are administered insub-therapeutic dosages.

In some embodiments, the two therapeutic agents are administeredsimultaneously.

In some embodiments, the two therapeutic agents are administeredsequentially.

In still another aspect, the invention provides a method for preventingor treating thrombosis and/or a thrombosis-related condition in a mammalcomprising administering to said mammal a therapeutically effectiveamount of the following three therapeutic agents:

-   -   (1)        [4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,        or a pharmaceutically acceptable salt thereof,    -   (2) another antiplatelet agent; and    -   (3) an anticoagulant agent.

In some embodiments, the antiplatelet agent is a cyclooxygenaseinhibitor. In some embodiments, the antiplatelet agent isacetylsalicylic acid. In other embodiments, the antiplatelet agent is anantagonist of TP receptor. In some embodiments, it is ifetroban. In someembodiments, the anticoagulant agent is a factor Xa inhibitor. In someembodiments, it is betrixaban.

In some embodiments, at least one of the therapeutic agents isadministered in a sub-therapeutic dosage. In some embodiments, all ofthe therapeutic agents are administered in sub-therapeutic dosages.

In some embodiments, the three therapeutic agents are administeredsimultaneously. In some embodiments, the three therapeutic agents areadministered sequentially.

In some embodiments, the pharmaceutically acceptable salt of[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureais the potassium salt.

In some embodiments, the pharmaceutically acceptable salt of[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureais the sodium salt.

In some embodiments, the thrombosis-related condition is selected fromthe group consisting of acute myocardial infarction, unstable angina,chronic stable angina, transient ischemic attacks, strokes, peripheralvascular disease, preeclampsia/eclampsia, deep venous thrombosis,embolism, disseminated intravascular coagulation and thromboticcytopenic purpura, thrombotic and restenotic complications followinginvasive procedures resulting from angioplasty, carotid endarterectomy,post CABG (Coronary artery bypass graft) surgery, vascular graftsurgery, stent placements and insertion of endovascular devices andprosthesis.

b. Pharmaceutical Compositions

This invention also provides pharmaceutical compositions comprising apharmaceutically acceptable carrier,[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof and at least anothertherapeutic agent selected from the group consisting of an anticoagulantagent, an antiplatelet agent, and combinations thereof.

In one aspect, the invention provides a pharmaceutical compositioncomprising the following two therapeutic agents:

-   -   (1)        [4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,        or a pharmaceutically acceptable salt thereof, and    -   (2) an anticoagulant agent;    -   and a pharmaceutically acceptable carrier.

In some embodiments, the anticoagulant agent is an inhibitor of factorXa.

In some embodiments, the factor Xa inhibitor is a specific factor Xainhibitor.

In some embodiments, the factor Xa inhibitor is YM-150, Daiichi DU-176b,LY517717, or a compound selected from Table 1.

In some embodiments, the factor Xa inhibitor is rivaroxaban.

In some embodiments, the specific inhibitor of factor Xa is betrixaban,or a pharmaceutically acceptable salt thereof. In still someembodiments, the pharmaceutically acceptable salt of betrixaban is themaleate salt.

In other embodiments, the anticoagulant is selected from the groupconsisting of specific inhibitors of thrombin, factor IXa, factor XI,factor XIa or factor VIIa. In some embodiments, the anticoagulant agentis selected from the group consisting of bivalirudin, argatroban,lepirudin, warfarin, and phenocoumarol.

In other embodiments, the anticoagulant agent is an injectableanticoagulant agent.

In some embodiments, the anticoagulant agent is selected from the groupconsisting of synthetic pentasaccharides, and low molecular weightheparin.

In some embodiments, the anticoagulant agent is selected from the groupconsisting of fondaparinux, danaparoid, enoxaparin, dalteparin andunfractionated heparin.

In other embodiments, the anticoagulant agent is an inhibitor of factorXI. In some embodiments, the inhibitor of factor XI is an anti-factor XIantibody.

In still other embodiments, the anticoagulant agent is bivalirudin.

In still some embodiments, the anticoagulant is ximelagatran, AZD0837 ordabigatran.

In another aspect, the invention provides a pharmaceutical compositioncomprising the following two therapeutic agents:

-   -   (1)        [4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,        or a pharmaceutically acceptable salt thereof, and    -   (2) another antiplatelet agent;    -   and a pharmaceutically acceptable carrier.

In some embodiments, the antiplatelet agent is an antagonist of TPreceptor. In some embodiments, the antagonist of TP receptor isifetroban.

In other embodiments, the antiplatelet agent is a cyclooxygenaseinhibitor. In some embodiments, the cyclooxygenase inhibitor isacetylsalicylic acid. In some embodiments, the antiplatelet agent is areversible cyclooxygenase-1 inhibitor. In some embodiments, thereversible cyclooxygenase-1 inhibitor is resveratrol.

In some embodiments, the antiplatelet agent is selected from the groupconsisting of abciximab, eptifibatide, tirofiban, dipyridamole,aggrenox, cilostazol, isbogrel, furegrelate and ozagrel.

In still another aspect, the invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and thefollowing three therapeutic agents:

-   -   (1)        [4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,        or a pharmaceutically acceptable salt thereof;    -   (2) another antiplatelet agent; and    -   (3) an anticoagulant agent.

In some embodiments, the antiplatelet agent is a cyclooxygenaseinhibitor. In some embodiments, the antiplatelet agent isacetylsalicylic acid. In other embodiments, the antiplatelet agent is anantagonist of TP receptor. In some embodiments, it is ifetroban. In someembodiments, the anticoagulant agent is a factor Xa inhibitor. In someembodiments, it is betrixaban.

In some embodiments, at least one of the therapeutic agents is presentin a sub-therapeutic dosage.

In some embodiments, all of the therapeutic agents are present insub-therapeutic dosages.

In some embodiments, the pharmaceutically acceptable salt of[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureais the potassium salt.

In some embodiments, the pharmaceutically acceptable salt of[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureais the sodium salt.

c. Kit

This invention also provides novel kits comprising

(1) a first container, wherein said first container contains[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof, and

(2) a second container, wherein said second container contains a secondtherapeutic agent selected from the group consisting of an anticoagulantagent, an antiplatelet agent, and a combination thereof.

In one aspect, the invention provides a kit comprising:

(1) a first container, wherein said first container contains[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof, and

(2) a second container, wherein said second container contains ananticoagulant agent.

In still another aspect, the invention provides a kit comprising:

(1) a first container, wherein said first container contains[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof, and

(2) a second container, wherein said second container contains ananother antiplatelet agent.

In some embodiments, at least one of the therapeutic agents is presentin a sub-therapeutic dosage.

In some embodiments, both of the therapeutic agents are present insub-therapeutic dosages.

In some embodiments, the kit further comprises a package insert statingthat the two therapeutic agents can be used together.

In still another aspect, the invention provides a kit comprising:

(1) a first container, wherein said first container contains[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof;

(2) a second container, wherein said second container contains ananticoagulant agent; and

(3) a third container, wherein said third container contains an anotherantiplatelet agent.

In some embodiments, the first container contains[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureapotassium salt.

In some embodiments, the first container contains[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureasodium salt.

In some embodiments, the second container contains betrixaban, or apharmaceutically acceptable salt thereof.

In still some embodiments, the second container contains betrixabanmaleate salt.

III. COMBINATION THERAPY

It is contemplated that a combination of Compound A with a factor Xainhibitor, such as betrixaban, will produce additional antithromboticeffect over the two agents alone. Example 3 shows that addition ofvarying concentrations of betrixaban to 1.1 μM of Compound A providedadditional thrombosis inhibition in a dose responsive manner in aperfusion chamber assay. Similarly, as shown by Example 4, addition ofvarying amount of Compound A to a fixed amount of betrixaban alsoproduced additional inhibition of thrombosis formation in a doseresponsive manner. Similar additive results were obtained in aplatelet-initiated thrombin generation assay as shown in Example 5,where the combination of Compound A and betrixaban provided greaterinhibition than either compound alone.

Inhibitors of other coagulation enzymes, such as factor XI inhibitors ordirect thrombin inhibitors, may also be combined with Compound A toachieve improved antithrombotic efficacy. Example 6 illustrates that thecombination of an antibody of factor XI with Compound A was capable ofinhibiting thrombus formation in assay conditions where neither CompoundA nor the factor XI antibody alone was able to produce detectableinhibition of thrombus formation. Example 7 illustrates that combinationof Compound A with a factor XI antibody produced more inhibition ofthrombus formation than Compound A alone or a combination of Compound Aand betrixaban in a perfusion assay. Example 8 illustrates the combinedantithrombotic effect observed when Compound A is combined with a directthrombin inhibitor, such as bivalirudin (Angiomax®).

Not only can Compound A provide additive antithrombotic benefit with ananticoagulant agent, it is also contemplated that Compound A, anantiplatelet agent acting through P2Y₁₂ antagonism, can be combined withother classes of antiplatelet agents to produce additionalantithrombotic benefit. As shown in Examples 9 and 10, additionalantithrombotic benefit was obtained when Compound A was combined witheither a cyclooxygenase inhibitor, such as acetylsalicylic acid (Example9), or a TP antagonist, such as ifetroban (Example 10), in the presenceof a factor Xa inhibitor.

It is contemplated that the method of treatment using a combination ofCompound A and a co-administered agent will not produce undesireddrug-drug interaction or other additional side effects over the agentsalone. Preferably, the combination can offer an improved efficacy and/orsafety advantage over the agents alone, particularly when smaller dosingis required to achieve a therapeutic result. In such a case, thetherapeutically effective amount of the agents in the combinationtherapy may be lower than the effective or optimal amount needed whenthe agents are used alone. It is contemplated that lower dosages willminimize potential side effects of an agent, thus lead to improvedsafety profile. Thus, the combination preferably allows one of thetherapeutic agents to be used at a sub-therapeutic dosage. Still morepreferably, the combination allows both therapeutic agents to be used atsub-therapeutic dosages.

Similarly, it is contemplated that the method of treatment using acombination of Compound A, an anticoagulant agent and anotherantiplatelet agent will not produce undesired drug-drug interaction orother additional side effects over use of any of the agents alone.Preferably the combination of three agents can offer an efficacy orsafety advantage over the use of any of the agents alone. Morepreferably the combination allows one of the therapeutic agents be usedat lower doses than that is required when the therapeutic agent is usedalone, i.e. at sub-therapeutic dosages. Still more preferably, thecombination allows all therapeutic agents to be used at sub-therapeuticdosages.

Compound A and the co-administered agent may be formulated into twoseparate pharmaceutical compositions. They may be administered at thesame time or sequentially in any order. Preferably, when administeredsequentially, the two agents are administered sufficiently closely intime so that the desired therapeutic effect can be provided. Compound Aand the co-administered agent may also be formulated into a singlepharmaceutical composition. Compound A, an anticoagulant agent andanother antiplatelet agent may also be administered at the same time orsequentially in any order. Preferably, when administered sequentially,the three agents are administered sufficiently closely in time so thatthe desired therapeutic effect can be provided. They may also beformulated into a single pharmaceutical composition or any two of themmay be formulated into a single pharmaceutical composition.

Any of the above dosage forms containing effective amounts are withinthe bounds of routine experimentation and within the scope of theinvention. A therapeutically effective dose may vary depending upon theroute of administration and dosage form. The preferred combination ofthe invention is a formulation that exhibits a high therapeutic index.The therapeutic index is the dose ratio between toxic and therapeuticeffects which can be expressed as the ratio between LD₅₀ and ED₅₀. TheLD₅₀ is the dose lethal to 50% of the population and the ED₅₀ is thedose therapeutically effective in 50% of the population. The LD₅₀ andED₅₀ are determined by standard pharmaceutical procedures in animal cellcultures or experimental animals. Combination therapies of thisinvention may be administered once or several times daily and otherdosage regimens may also be useful. Preferably, combination therapies ofthis invention are administered in a single daily dose, or administeredtwo, three, or four times daily. More preferably, combination therapiesof this invention are administered once or twice daily.

Typically, about 0.5 to 500 mg of Compound A, or a salt or mixture ofsalts of Compound A is compounded with a physiologically acceptablevehicle, carrier, excipient, binder, preservative, stabilizer, dye,flavor etc., as called for by accepted pharmaceutical practice. In oneaspect, Compound A is formulated into a formulation suitable forintravenous administration. In some embodiments, a unit dose of theintravenous formulation contains from 1 to 50 mg of Compound A or apharmaceutically acceptable salt. In other embodiments, the unit dosecontains from 5 to 40 mg, 10 to 30 mg, 15 to 25 mg, 25 to 45 mg, orabout 20 mg, 30, 40, or 50 mg of Compound A or the salt.

In another aspect, Compound A is formulated into a formulation suitablefor oral administration. In some embodiments, the composition isformulated as a unit dose containing from 1 to 800 mg, 20 to 200 mg, 50to 150 mg, 10 to 50 mg, or 20 to 40 mg of Compound A or a salt. In someembodiments, the composition is in a unit dose format and contains about30, 50, 75, 100, 125, 150, 175, or 200 mg of Compound A or a salt.

When Compound A and the co-administered agent are formulated into asingle pharmaceutical composition, about 0.5 to 500 mg of theco-administered agent can be added to the above composition. Preferably,when Compound A and the other agent are formulated in an intravenousformulation, Compound A or a salt thereof is present in the amount of 1to 50 mg, 5 to 40 mg, 10 to 30 mg, 15 to 25 mg, 25 to 45 mg, or about 20mg, 30, 40, or 50 mg. When Compound A and the other agent are formulatedin an oral formulation, Compound A or a salt is present in the amount offrom 1 to 800 mg, 20 to 200 mg, 50 to 150 mg, 10 to 50 mg, or 20 to 40mg or about 30, 50, 75, 100, 125, 150, 175, or 200 mg. In combinationscontaining Compound A and betrixaban, any of the above unit doses ofCompound A or a salt or mixture of salts of Compound A and about 0.5 to500 mg of betrixaban or a salt or mixture of salts of betrixaban arecompounded with a physiologically acceptable vehicle, carrier,excipient, binder, preservative, stabilizer, dye, flavor etc., as calledfor by accepted pharmaceutical practice. The amount of activeingredient(s) in these compositions is such that a suitable dosage inthe range indicated is obtained.

It is contemplated that a typical dosage of Compound A in thecombination therapies will range from about 0.001 mg/kg to about 100mg/kg, preferably about 0.01 mg/kg to about 11.4 mg/kg, more preferablyfrom about 0.01 mg/kg to about 2.85 mg/kg, and even more preferably fromabout 0.01 mg/kg to about 1.43 mg/kg. In combination therapiescontaining Compound A and betrixaban, it is contemplated that a typicaldosage of betrixaban will range from about 0.001 mg/kg to about 1000mg/kg, preferably from about 0.01 mg/kg to about 2.0 mg/kg, and morepreferably from about 0.1 mg/kg to about 1.5 mg/kg, or from about 0.4mg/kg to about 1.2 mg/kg, and even more preferably from about 0.5 mg/kgto about 1.0 mg/kg. Still more preferably, the dosage of betrixaban inthe combinations is lower than 0.5 mg/kg.

The typical dosages of the other co-administered agents described hereinwhen used as a single agent are known to a person skilled in the art. Itis contemplated that the dosages of these agents when used incombination with Compound A will not exceed the maximum dosages of theindividual agents. Preferably, the dosages in the combination therapiesare less than the maximum dosages and more preferably, the dosages inthe combination therapies are sub-therapeutic dosages. It iscontemplated that the dosages can be adjusted to reflect the improvedbenefit achieved by the combination therapies, which can be determinedby one skilled in the art based on the information given herein.

IV. COMPOSITION

The present invention further provides a novel composition comprisingCompound A or a pharmaceutically acceptable salt thereof, ananticoagulant agent or another antiplatelet agent, and apharmaceutically acceptable carrier.

The pharmaceutical compositions of the invention can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders, tablets,capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions. Formulations may optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these.

Pharmaceutical formulations may be prepared as liquid suspensions orsolutions using a sterile liquid, such as oil, water, alcohol, andcombinations thereof. Pharmaceutically suitable surfactants, suspendingagents or emulsifying agents, may be added for oral or parenteraladministration. Suspensions may include oils, such as peanut oil, sesameoil, cottonseed oil, corn oil and olive oil. Suspension preparation mayalso contain esters of fatty acids, such as ethyl oleate, isopropylmyristate, fatty acid glycerides and acetylated fatty acid glycerides.Suspension formulations may include alcohols, such as ethanol, isopropylalcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, suchas poly(ethyleneglycol), petroleum hydrocarbons, such as mineral oil andpetrolatum, and water may also be used in suspension formulations.

The compositions of this invention are formulated for pharmaceuticaladministration to a mammal, preferably a human being. Suchpharmaceutical compositions of the invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally orintravenously. The formulations of the invention may be designed asshort-acting, fast-releasing, long-acting, sustained-releasing. Stillfurther, compounds can be administered in a local rather than systemicmeans, such as administration (e.g., injection) as a sustained releaseformulation.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation. Compounds may be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection may be in ampoules or inmulti-dose containers.

The pharmaceutical compositions of this invention may be in any orallyacceptable dosage form, including capsules, tablets, aqueous suspensionsor solutions. In the case of tablets for oral use, carriers that arecommonly used include lactose and corn starch. Lubricating agents, suchas magnesium stearate, are also typically added. For a capsule form,useful diluents include lactose and dried cornstarch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may bein the form of suppositories for rectal administration. These may beprepared by mixing the agent with a suitable non-irritating excipientwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also be in atopical form, especially when the target of treatment includes areas ororgans readily accessible by topical application, including diseases ofthe eye, the skin, or the lower intestinal tract. Suitable topicalformulations are readily prepared for each of these areas or organs.

Topical application for the lower intestinal tract may be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used. For topicalapplications, the pharmaceutical compositions may be formulated in asuitable ointment containing the active component suspended or dissolvedin one or more carriers. Carriers for topical administration of thecompounds of this invention include, but are not limited to, mineraloil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical compositions may be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters, wax, cetyl alcohol, 2-octyldodecanol, benzyl alcohol andwater.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative, such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment, such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons and/or other conventional solubilizing ordispersing agents.

In addition to dosage forms described above, pharmaceutically acceptableexcipients and carriers and dosage forms are generally known to thoseskilled in the art and are included in the invention. It should beunderstood that a specific dosage and treatment regimen for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health, sex and diet, renal and hepatic function of the patient,and the time of administration, rate of excretion, drug combination,judgment of the treating physician or veterinarian and severity of theparticular disease being treated. The amount of active ingredients willalso depend upon the therapeutic agent combined with Compound A.

V. KIT OF PARTS

The invention further provides a novel kit or package. In someembodiments, the kit of the present invention comprises: (a) a firstcontainer containing Compound A or pharmaceutically acceptable saltforms thereof, and (b) a second container containing an anticoagulantagent or another antiplatelet agent. In other embodiments, the kitcomprises: (a) a first container containing Compound A orpharmaceutically acceptable salt forms thereof, (b) a second containercontaining an anticoagulant agent and (c) a third container containinganother antiplatelet agent. In some embodiments, the kit furthercontains a package insert stating that the two pharmaceutical agents canbe used together for the treatment of a condition characterized byundesired thrombosis.

The first, second, or third container can be a bottle, jar, vial, flask,syringe, tube, bag, or any other container used in the manufacture,storage, or distribution of a pharmaceutical product. The package insertcan be a label, tag, marker, or the like, that recites informationrelating to the pharmaceutical composition of the kit. The informationrecited will usually be determined by the regulatory agency governingthe area in which the pharmaceutical composition is to be sold, such asthe United States Food and Drug Administration. Preferably, the packageinsert specifically recites the indications for which the pharmaceuticalcomposition has been approved. The package insert may be made of anymaterial on which a person can read information contained therein orthereon. Preferably, the package insert is a printable material, such aspaper, adhesive-backed paper cardboard, foil, or plastic, and the like,on which the desired information has been printed or applied.

VI. EXAMPLES

Unless stated otherwise, the abbreviations used throughout thespecification have the following meanings:

-   -   ACN=acetonitrile    -   API=active pharmaceutical ingredient    -   aq.=aqueous    -   Boc=tert-butoxylcarbonyl    -   DCM=dichloromethane    -   DMSO=dimethyl sulfoxide    -   eq.=equivalent    -   EtOH=ethanol    -   g=gram    -   HPLC=high performance liquid chromatography    -   hr=hour    -   kg=kilogram    -   KOH=potassium hydroxide    -   L=liter    -   LOD=limit of detection    -   M=molar    -   Me=methyl    -   MeO=methoxy    -   MeOH=methanol    -   mg=milligram    -   min=minute    -   mL=milliliter    -   mm=millimeter    -   N=normal    -   ng=nanogram    -   nM=nanomolar    -   NMR=nuclear magnetic resonance    -   pg=picogram    -   pM=picomolar psi pound per square inch    -   sec=second    -   THF=tetrahydrofuran    -   TLC=thin layer chromatography    -   WFI=water for injection    -   μM=micromolar    -   μg=microgram    -   Z-gly-gly-arg-AMC=carbobenzyloxy-glycine-glycine-arginine-4-aminomethylcoumarin    -   VC=vehicle control    -   NS=nonsignificant

Example 1 Preparation of Compound A and Its Potassium Salt Formula I

Step 1:

Methyl 2-amino-4,5-difluorobenzoate (1) (38 kg, 1.0 eq.) anddichloromethane (560 kg, 8×, ACS>99.5%) were charged to a 2000 L GLreactor. The reaction mixture was agitated for mins.4-Nitrophenylchloroformate (49.1 kg, 1.2 eq.) was charged into the 200 Lreactor followed by dichloromethane (185 kg) and the contents wereagitated for 5 mins. After pressurizing the 200 L reactor, the4-nitrophenylchloroformate solution was transferred into a 2000 Lreactor containing dichloromethane solution of compound 1. The reactionmixture was heated to 40±5° C. (reflux) under nitrogen gas purge for 3hrs. Representative TLC analysis confirmed completion of reaction(in-process TLC, no compound 1 remaining; 99:1 CHCl₃-MeOH). The solutionwas cooled to 30° C. and 460 kg of dichloromethane was distilled offunder vacuum. The 2000 L reactor was charged with 520 kg of hexanes andthe contents of the reactor were cooled to 0±5° C. and agitated for 4hrs. The solid obtained was filtered through a GF Nutsche filter linedwith a sheet of T-515 LF Typar filter and a sheet of MeI-Tuf 1149-12filter paper. The filter cake was washed with 20 kg of hexanes andvacuum dried at 35° C. until constant weight was attained. The dryproduct was discharged (70.15 kg) with 98% yield. The product 2 wasconfirmed by ¹H NMR and TLC analysis.

Step 2:

A 2000 L GL reactor was charged with compound 2 (64.4 kg, 1.0 eq.),anhydrous tetrahydrofuran (557 kg) and triethylamine (2.2 kg, 0.1 eq.).The charging line of the 2000L GL reactor was rinsed withtetrahydrofuran (10 kg). The contents of the reactor were agitated for25 mins, during which period a complete solution was obtained. A 200L HPreactor was charged with N-Boc-p-phenylenediamine (38 kg, 1.0 eq.),tetrahydrofuran (89 kg) and agitated for 30 mins until a completesolution was obtained. The contents of the 200 L HP reactor weretransferred to the 2000 L GL reactor containing compound 2 and thenheated at 65±5° C. for 2 hrs. The reaction was deemed complete by HPLCafter confirming the disappearance of starting material 2 when theamount of compound 2 remaining in the reaction mixture is <1%.

The contents of the 2000 L GL reactor were cooled to 20±5° C. and thencharged with sodium methoxide (25% solution in methanol, 41.5 kg, 1.05eq.) over 20 mins, while maintaining the temperature below 30° C. Thecharging lines were rinsed with tetrahydrofuran (10 kg). The contentswere agitated at 25±5° C. for 4 hrs. In-process HPLC analysis confirmedthe completion of the reaction when the amount of compound 3a remainingin the reaction mixture is <1%. To this reaction mixture was addedfiltered process water (500 kg) and the contents of the 2000L GL reactorwere distilled under vacuum into a clean 200 L GL receiver until 300 kgof solvent was distilled. The solids obtained were filtered using a GLNutsche filter and washed with process filtered water until the color ofthe solid 3b was white to grayish.

The 2000 L GL reactor was charged with wet compound 3b filter cake,dioxane (340 kg) and the contents were agitated for 1 hr. The filterablesolid obtained were filtered through a GL Nutsche filter with a sheet ofT-515 LF Typar filter paper. The solid cake was blow dried for 2 hrs andthen charged with dioxane (200 kg) into the 2000 L GL reactor. Thecontents were agitated for 10 mins and then charged with 4 N HCl indioxane (914 kg) over 3 hrs while the internal temperature wasmaintained at below 30° C. The charging line was rinsed with additionaldioxane (10 kg) and the contents of the reactor were agitated for 6 hrsat 25±5° C. The completion of the reaction was monitored by HPLC for theconversion of compound 3b to compound 3 (in process control showscompound 3b is <1% in the reaction mixture). The contents of the reactorwere cooled to 5+5° C. for 2 hr and the solid obtained was filteredthrough a GL Nutsche filter followed by washing with dioxane (50 kg).The filter cake was blow-dried with 8±7 psi of nitrogen for 30 mins andthe purity was analyzed by HPLC. The filtered solid was dried to aconstant weight in vacuum oven at 45° C. for 48 hrs. The compound 3(65.8 kg, actual yield 110.6%) was discharged and analyzed by ¹HNMR andHPLC. ¹H NMR (DMSO): δ 11.75 (s, 1H), 7.88 (dd, 1H), 7.32 (m, 4H), 7.21(dd, 1H).

Step 3:

A 200 L HP reactor was charged with compound 3 (18 kg, 1.0 eq.) andpressurized with 100±5 psi of nitrogen. The nitrogen from the reactorwas vented through the atmospheric vent line and the condenser valve wasopened. Dimethyl sulfoxide was then charged into the reactor (>99.7%,105 kg) under blanket of argon. The reactor contents were agitated at22° C. (19-25° C.) for 15 mins and then maximum achievable vacuum waspulled on the 200 L HP reactor and all valves were closed. Using theestablished vacuum methylamine (33 wt % in absolute ethanol, 37.2 kg)was charged to the 200 L HP reactor at a rate that maintains theinternal temperature at 25±5° C. A nitrogen blanket on the reagentsolution was maintained during charging. After the charging line wasrinsed with dimethyl sulfoxide (5 kg), the 200 L HP reactor condenservalve was closed and the reactor contents were heated to 110±5° C. Thecontents of the reactor were agitated for at least 5 hrs at 110±5° C.In-process HPLC taken after 5 hr 40 mins showed compound 3 content of0.09%, indicating completion of the reaction (in-process specificationrequires amount of compound 3<1%). The contents of the 200 L HP reactorwere cooled to 25×5° C. While the 200 L reactor was cooling, all thevalves of a 2000 L GL reactor were closed and process filtered water(550 kg) was charged. The contents of the 200 L HP reactor weretransferred to the 2000 L GL reactor over 15 mins followed by rinsingthe charging line with process filtered water (50 kg). The contents ofthe 2000 L GL reactor were agitated for 2 hrs at 5±5° C. The filterablesolids obtained were filtered onto a GL nutsche filter fitted withMeI-Tuf 1149-12 filter paper under vacuum. The wet filter cake wasdischarged and transferred into pre-lined vacuum trays with Dupont'sfluorocarbon film (Kind 100A) and special oven paper (KAVON 992) andtransferred to the vacuum oven tray dryer. The oven temperature was setto 55° C. and compound 4 was dried for 12 hrs to a constant weight. Theproduct 4 was discharged (12.70 kg) in 76.5% yield (expected 85-95%).HPLC shows 98.96% purity and ¹H NMR confirmed the structure for compound4. ¹H NMR (DMSO): δ 11.10 (s, 1H), 7.36 (d, 1H), 6.78 (d, 2H), 6.75 (m,1H), 6.56 (d, 2H), 6.20 (d, 1H), 5.18 (d, 2H), 2.76 (d, 3H).

Step 4:

A 200 L HP reactor was charged with compound 4 (20.7 kg, 1.0 eq.), ethyl5-chlorothiophene-2-ylsulfonylcarbamate (37.5 kg, 2.0 eq. >95%),dimethyl sulfoxide (>99%, 75 kg) and agitated for 15 mins. Maximumachievable vacuum was pulled and the 200 L HP reactor was heated at65±5° C. for 15 hrs. In-process HPLC analysis of the representativesample from the reactor indicated <0.9% compound 4 remaining in thereaction mixture (in-process criteria for reaction completion iscompound 4<1%). A 800 L reactor was charged with process filtered water(650 kg) and then the contents of the 200 L HP reactor were transferredto the 800 L reactor while the internal temperature was maintained below25° C. The 200 L HP reactor was rinsed with dimethyl sulfoxide (15 kg)which was transferred to the 800 L reactor which was then agitated for 2hrs at 5±5° C. The solid formed was filtered through a filter to a 200 LGL receiver under vacuum and the filter cake was rinsed with processfiltered water (60 kg). HPLC analysis of a representative sample of thewet cake showed the purity of Compound A was <95%, indicatingdichloromethane trituration was needed based on in-process control. The800 L GL reactor was charged with the wet Compound A, dichloromethane(315 kg) and the contents were agitated for 3 hrs. The solid wasfiltered through GL nutsche filter lined with 1 sheet of T515 LF TYPARfilter under vacuum. The filter cake was washed with dichloromethane (50kg) and the cake was blow dried with 8±7 psi of nitrogen for 15 mins.The filter cake was transferred into pre-lined vacuum trays with Dupontfluorocarbon film (Kind 100A) and then dried in the vacuum oven traydryer at 60° C. for 12 hrs. The dried Compound A was isolated (33.6 kg,93% yield) with HPLC purity of 93.5% and 4.3% of sulfonamide. ¹H NMRconfirmed the structure for Compound A. ¹H NMR (DMSO): δ 11.20 (s, 1H),9.15 (s, 1H), 7.68 (d, 1H), 7.42 (d, 2H), 7.36 (d, 1H), 7.26 (m, 1H),7.16 (d, 2H), 6.78 (m, 1H), 6.24 (d, 1H), 2.78 (d, 3H).

Step 5:

A 800 L GL reactor was charged with acetonitrile (134 kg), WFI qualitywater (156 kg) and the contents was agitated for 5 mins. To this thencharged Compound A (33.6 kg, 1.0 eq.) and the reaction mixture was asuspension at this point. The suspension was charged with aqueoussolution (WFI water, 35 kg) of potassium hydroxide (4.14 kg, 1.15eq., >85%) at a rate that maintained the internal temperature at below30° C. The charging lines were rinsed with WFI quality water (2 kg) andthe 800 L GL reactor contents were heated to 50±5° C. for 1 hr. Thecontents were then filtered hot through a bag filter, then a sevencartridge 0.2μ polish filter to clean HDPE drums. The hot filtrationsystem was maintained through out the filtration process so no materialcrashed out of the solution. The 800 L GL reactor jacket was cooled to25±5° C. before the 800L GL reactor was rinsed with a pre-mixed solutionof acetonitrile (8.5 kg) and WFI quality water (10 kg) through thefilter system into the drums labeled as Compound A hot filtration. Usingthe pressure vessel the 800L GL reactor was rinsed with WFI qualitywater (20 kg) followed by acetone (20 kg) then blow dried with nitrogen(3+2 psi). The 800 L GL reactor bottom valve was closed and 20+10 inchesHg of vacuum was pulled, then the contents of the drums labeled asCompound A hot filtration was charged to the reactor. The 800 L GLreactor contents were cooled to 20±5° C. and then using a polish filter,the reactor was charged with methanol (373 kg, >99%) while maintainingthe internal temperature below 30° C. The contents of the 800 L GLreactor were cooled to 15±5° C. followed by agitation of the contentsfor 12 hrs at this temperature. During this time the filterable solidswere filtered through a clean filter apparatus into a clean 200 L GLreceiver followed by pressurizing the reactor, pulling 20+10 inches Hgof vacuum on the filter/receiver and filtered the contents. The filtercake was washed with methanol (30 kg) and blow dried with 8+7 psi ofnitrogen for 10 mins. The vacuum oven tray dryer temperature was set to80° C. prior to loading the wet cake of the salt Formula I. The wetfilter cake was transferred into the pre-lined vacuum trays withDupont's fluorocarbon film (Kind 100A) and the special oven paper (KavonMeI Tuf paper) and dried in the vacuum oven tray dryer at an oventemperature of 80° C. to a constant weight (constant weight is definedas tray reading at least 1 hr apart having the same weight within +50g). A representative sample was analyzed (residual solventspecifications for API) and showed that residual solvents met thespecifications. The final API was subjected to equilibration with water(5-6%) for 12 hrs with a tray of WFI quality water present, thenthoroughly turned and allowed to stand for an additional 12 hrs andfinally subjected to KF (Karl Fischer) water analysis (5.5% watercontent). The salt Formula I was transferred (21.80 kg, 60.6% yield) todouble heavy-duty poly bags and stored in secondary containment. HPLCshowed purity of 99.7% for and ¹H NMR confirmed the structure ofCompound A. ¹H NMR (DMSO): δ 11.14 (s, 1H), 8.60 (s, 1H), 7.48 (m, 2H),7.35 (d, 1H), 7.22 (d, 1H), 6.95 (m, 3H), 6.75 (m, 1H), 6.22 (d, 1H),2.78 (d, 3H).

Additional methods of preparation and analysis of Compound A and itssalt forms are described in U.S. Patent Application Publication US2007/0123547, filed Nov. 3, 2006, the contents of which are incorporatedherein by reference in its entirety.

Example 2 Preparation of Betrixaban

Step 1:

5-Methoxy-2-nitrobenzoic acid (5) (25.0 kg, 1.0 eq.),2-amino-5-chloropyridine (6) (16.3 kg, 1.0 eq.), and acetonitrile (87.5kg, 3.5 parts) were charged to a 380 L GLMS reactor. The reactionmixture was adjusted to 22° C. (19 to 25° C.) and anhydrous pyridine(30.0 kg, 3.0 eq.) was added. The pump and lines were rinsed forwardwith acetonitrile (22.5 kg, 0.9 parts), and the reactor contents wereadjusted to a temperature of 19-22° C. Phosphorous oxychloride (23.3 kg,1.20 eq.) was charged to the contents of the reactor via a meteringpump, while maintaining a temperature of 25° C. (22-28° C.). Themetering pump and lines were rinsed forward with acetonitrile (12.5 kg,0.5 parts), while keeping the temperature at 25° C. (22-28° C.). Thereaction mixture normally turned from a slurry to a clear solution afterthe addition of about ⅓ of the POCl₃. At the end of the addition, itbecame turbid. After complete addition, the reaction mixture wasagitated at 25° C. (22-28° C.) for ca. 1 hr, at which time HPLC analysisconfirmed reaction completion. The solution was cooled to 15° C. (12-18°C.) and drinking water (156.3 kg, 6.25 parts) was charged slowly whilekeeping reaction temperature between 12 and 30° C. The reaction mixturewas then adjusted to 22° C. (19 to 25° C.) and agitated for ca. 5 hrsuntil exotherm ceased. Formation of a slurry was visually confirmed andthe contents of the reactor were filtered onto a pressure nutsche fittedwith a filter cloth. The reactor, pump, and lines were washed forwardonto the pressure nutsche with two portions of drinking water (62.5 kg,2.5 parts each). The filtrate had a pH value of 7. The product (41.8 kg)was dried under vacuum with a maximum temperature of water bath (to heatdryer jacket) of 50° C. After ca. 12 hrs, in-process LOD analysisindicated a solvent content of 0.72%. The dry product 7 was discharged(34.4 kg) with 88.2% yield and 99.1% purity by HPLC.

Step 2:

To a 780 L Hastelloy reactor, compound 7 (33 kg, 1.0 eq), 5% platinumcarbon (sulfided, 0.33 kg, 0.010 parts) and dichloromethane (578 kg,17.5 parts) were charged. Agitation was started and reactor contentswere adjusted to 22° C. (19 to 25° C.). The reactor was pressurized withca. 30 psi hydrogen and the reaction mixture gently heated to 28° C.(25-31° C.). Hydrogenation of the reactor contents was performed underca. 30 psi at 28° C. (25 to 31° C.; maximum 31° C.) until the reactionwas complete by HPLC. After 16.5 hrs, the reaction was deemed completeafter confirming the disappearance of starting material (0.472%). Thecontents of the reactor were circulated through a conditioned celite pad(0.2-0.5 kg celite conditioned with 20-55 kg dichloromethane) preparedin a 8″ sparkler filter to remove the platinum catalyst. The reactor andcelite bed were rinsed forward with two portions of dichloromethane (83kg, 2.5 parts each). The filtrate was transferred to and concentrated ina 570 L GLMS reactor under a atmospheric pressure to ca. 132 L (4 partsvolume). Ethanol (69 kg, 2.1 parts) was charged and concentrationcontinued under atmospheric pressure to ca. 99 L (3 parts volume).In-process NMR indicated that the dichloromethane content was 39%.Ethanol (69 kg, 2.1 parts) was charged again and concentration continuedagain to ca. 99 L (3 parts volume). In-process NMR indicated that thedichloromethane content was 5%. The reaction mixture was then adjustedto 3° C. (0 to 6° C.), agitated for ca. 1 hr, and the resulting slurryfiltered onto a jacketed pressure nutsche fitted with a filter cloth.The reactor, pump, and lines were rinsed forward with cold (3° C. (0-6°C.)) ethanol (26 kg, 0.8 parts). The wet filter cake (36.6 kg) was driedunder vacuum at 40-50° C. with a maximum temperature of water bath (toheat dryer jacket) of 50° C. LOD analysis after 12.5 hrs indicatedsolvent content was at 0.1%. The dry product 8 was discharged (26.4 kg)in 89.5% yield. HPLC showed 98.4% purity, with dechlorinated impurity at0.083%.

Step 3:

To a 780 L Hastelloy reactor, was charged 4-cyanobenzoyl chloride (9)(17.2 kg, 1.1 eq.) and THF (92 kg, 3.5 parts). Reactor contents wereagitated at 22° C. (19 to 25° C.) until all of the solids had dissolved.The resulting solution was transferred to a lower receiver and thereactor was rinsed forward with THF (26 kg, 1 part). Compound 8 (26.4kg, 1 eq.), THF (396 kg, 15 parts) and pyridine (2.90 kg, 0.4 eq.) werecharged to a clean reactor. The pump and lines were rinsed forward withTHF (34 kg, 1.3 parts). Via a metering pump, the 4-cyanobenzoylchloride/THF solution was charged to the reactor, keeping thetemperature at <30° C. and rinsing forward with THF (ca. 10 kg). Theresulting yellow-colored slurry was agitated at 22° C. (19 to 25° C.)for ca 2 hrs. In-process HPLC taken after 2 hrs showed a compound 8content of 0%, indicating completion of the reaction. The slurry wasfiltered onto a pressure nutsche fitted with a filter cloth. Thereactor, pump, lines, and wet cake were rinsed with three portions ofethanol (ca. 15 kg each). The wet filter cake was discharged (65.4 kg)and transferred back to the reactor for slurry wash in ethanol (317 kg,12 parts) at 22° C. (19 to 25° C.) for ca. 1 hr. The slurry was filteredonto the pressure nutsche and the reactor, pump, lines, and wet filtercake were rinsed with two portions of ethanol (ca. 15 kg each) and twoportions of THF (ca. 15 kg each). The wet filter cake was dried undervacuum with a maximum temperature of warm glycol bath (to heat thereactor jacket) of 40° C. After 14.5 hrs of drying, LOD was 0.75%. Thedried material was milled (screen 0.125″) to give 31.8 kg of compound10, which was dried under vacuum for another 10.5 hrs. LOD after dryingwas 1.8%, and the product was discharged (31.5 kg) in 74.8% yield(expected 60-90%). HPLC showed 100% purity.

Step 4:

A slurry of compound 10 (455 g, 1.0 eq.) in THF (4.67 kg, 10.3 parts)was prepared and adjusted to <10° C. Lithium dimethyl amide was preparedas follows: hexyllithium (2.3 N/hexane, 2.45 L, 5.5 eq.) was added todimethylamine solution (2 N/THF, 2.8 L, 5.5 eq.) maintaining <10° C. Thelithium dimethyl amide solution was charged into the slurry containingthe compound 10 keeping the pot temperature of <10° C. The reactionprogress was monitored by in-process HPLC which confirmed that theamount of compound 10 was <1.0%. A buffer solution of NaHCO₃ (490 g, 1.1parts, 5.7 eq.) and Na₂CO₃ (490 g, 1.1 parts, 4.5 eq.) in deionizedwater (6.6 kg, 14.51 parts) was prepared, and above reaction mixture wastransferred to this aqueous solution maintaining <5° C. The productprecipitated out and the resulting slurry was adjusted to 20° C. over aperiod of 12 hrs. The solid was filtered, and the resulting wet cake waswashed with 3.5 kg (7.7 parts) of deionized water. The solid wasfiltered off using a coarse frit glass bench filter, and rinsedforwarded with cold (0-5° C.) absolute ethanol (628 g, 1.4 parts). Theproduct betrixaban was dried at 30-35° C. Dry product was obtained in458 g (73% yield).

Example 3 Combination of Compound A and Betrixaban in a PerfusionChamber Thrombosis Assay (I)

The real time perfusion chamber assay couples the features of animalthrombosis models that use intravital microscopy to those of perfusionchamber technology in order to produce an assay suited to monitoringdrug activity in clinical trials. This assay perfuses whole bloodthrough capillaries at arterial rates of shear, exposing the blood tothrombogenic type III collagen. Platelets are labeled with a fluorescentdye (rhodamine 6 G) prior to perfusion such that analysis of thethrombus deposition can be performed by measurement of fluorescenceintensity inside the perfusion chamber. Quantification is performed byanalysis of the thrombus height (fluorescence intensity (number ofpixels)/total area (μm²)).

Compound A was tested in this assay at a concentration capable ofgenerating equivalent levels of inhibition of ADP induced plateletaggregation in human platelet rich plasma as targeted by clopidogrel, awidely used antiplatelet agent whose antithrombotic activity is mediatedby inhibition of the platelet P2Y₁₂ receptor. As shown in FIG. 1,treatment of blood with Compound A at 1.1 μM, while capable ofsignificant inhibition of platelet aggregation produced only partialinhibition of thrombosis (23%±14%, p>0.05, n=4). Addition of betrixabanto blood already treated with Compound A produced significant inhibitionof thrombosis (68%±3%, 59%±14%, and 57%±11% inhibition at 1 μM, 300 nMand 100 nM betrixaban respectively, p<0.01, n=4). FIG. 2 demonstratesthe dose dependent inhibition of thrombosis at betrixaban concentrationsbetween 1 μM and 3 nM.

Example 4 Combination of Compound A and Betrixaban in a Real-TimeThrombosis Assay (II)

Blood was collected by venipuncture from healthy volunteers into 5 μMbetrixaban. Increasing concentrations of Compound A were added in vitro.Following 20 mins incubation, blood was perfused through the collagencoated capillary (type III collagen, 1600 sec⁻¹). As seen in FIG. 3,increasing concentrations of Compound A demonstrated dose responsiveinhibition of thrombosis, with 10 μM of Compound A resulting in a singlemonolayer of platelets.

Example 5 Combination of Compound A and Betrixaban in a ThrombinGeneration Assay

Whole blood from healthy volunteer donors was collected by venipunctureand drawn into 3.2% trisodium citrate (Vacutainer, Becton Dickinson) foranticoagulation. Platelet rich plasma was prepared by centrifugation andthe platelet count was adjusted to 150,000/μL. In a 96 well plate,platelets were activated by addition of convulxin (200 ng/mL) andincubation at 37° C. for 3 mins. Subsequent to platelet activation,thrombin generation was initiated by addition of 23 pM tissue factor(Innovin, Dade Behring) and 15 mM calcium. Thrombin activity wasmonitored by cleavage of the specific fluorogenic substrate(Z-gly-gly-arg-AMC, Bachem) in a fluorescence plate reader.

In platelets derived from human donors, half maximal inhibition of ADPinduced platelet aggregation was achieved at a Compound A concentrationof 4.7±5.1 μM (n=19). For the factor Xa inhibitor betrixaban, aconcentration of 31.25 nM was adequate for equivalent inhibition ofthrombin generation as that achieved by a therapeutic anticoagulant(Pentasaccharide fondaparinux). As shown in FIG. 4, even when Compound Awas used at a concentration capable of maximal inhibition of plateletaggregation (10 μM), there was a substantial level of thrombingeneration in this ex vivo system. Similar results of thrombingeneration were obtained by using the factor Xa inhibitor betrixaban atits therapeutic concentration. However, a combination of the two agentsprovided greater inhibition of thrombin production. Since thrombinactivity is a mediator of thrombotic conditions, the combination of thetwo agents has the potential to produce superior activity than each ofthe single agents alone.

Example 6 Combination of Compound A and an Anti-Factor XI Antibody (I)

In this example, non-anticoagulated whole blood was collected fromhealthy volunteers, and rhodamine 6G (which fluorescently labelsplatelets), along with an antibody to factor XI (Hemetech) and CompoundA, before perfusing through a collagen-coated capillary (type Icollagen) at arterial shear rates (1000 s⁻¹). After perfusion the sizeof the platelet thrombus was measured by fluorescence. As shown in FIG.5, the anti-factor XI antibody alone did not reduce thrombus size at upto 10 μg/mL concentration under the assay conditions. Compound A alonehad no significant effect on platelet thrombus size at 10 μM in thisassay. However, when increasing concentrations of a factor XI antibodywere combined with 10 μM of Compound A, a dose proportional inhibitionof thrombus size was observed, reaching a maximum of 50% inhibition at10 μg/mL of the factor XI antibody.

Example 7 Combination of Compound A and an Anti-Factor XI Antibody (II)

Capillaries were coated with a combination of type I collagen and tissuefactor (1/100 ratio of collagen to Innovin (Dade-Behring).Non-anticoagulated blood was combined with rhodamine 6G, 10 μM ofCompound A and either betrixaban or an antibody against factor XI beforeperfusion through the capillary. As shown in FIG. 6, the combination ofCompound A and the factor XI antibody was able to produce significantlymore inhibition of thrombus formation than Compound A alone or thecombination of Compound A and betrixaban.

Example 8 Combination of Compound A and Bivalirudin, a Direct ThrombinInhibitor

As an example of additive antithrombotic benefit achieved when CompoundA was combined with a direct thrombin inhibitor, such as bivalirudin, inthe following assay, blood was collected in 12 μg/mL bivalirudin(standard therapeutic concentration), incubated with variousconcentrations of Compound A (0.1, 1 or 10 μM) for 20 mins in thepresence of rhodamine 6G (which fluorescently labels platelets),followed by perfusion through a glass capillary coated with collagen(type III) at a fixed shear rate (1600 s⁻¹). Under these conditions,bivalirudin alone did not significantly inhibit the thrombotic process;however, a dose-dependent inhibition of thrombosis was observed whenincreasing amounts of Compound A are present in combination withbivalirudin. FIG. 7 illustrates the combined antithrombotic benefitachieved by the combination of Compound A and bivalirudin.

Example 9 Combination of P2Y₁₂ Antagonist Compound A and Aspirin, aCyclooxygenase Inhibitor, and a Factor Xa Inhibitor

Using a real-time perfusion chamber assay to assess the thromboticprofile of healthy donors where human blood was collected byvenipuncture from aspirinated donors (81 mg or 325 mg of aspirin dailyfor 3 days), the additive antithrombotic benefit of Compound A on top ofaspirin was assessed. In this assay, glass capillaries were coated withtype I collagen, and human whole blood (incubated for 20 mins withrhodamine-6G, which fluorescently labels platelets) collected in afactor Xa inhibitor, C921-78 (see Betz A, Wong P W, Sinha U. Inhibitionof factor Xa by a peptidyl-alpha-ketothiazole involves 2 steps: evidencefor a stabilizing conformational change. Biochemistry 1999; 38:14582-14591, incorporated herein by reference in its entirety), wasperfused through the capillary at a fixed sheer rate (1600 sec⁻¹).During perfusion, the extent of thrombosis was quantified by measurementof mean fluorescence intensity/area (μm²), which is a measure ofplatelet deposition on the collagen surface.

As shown in FIG. 8, increasing concentrations of Compound A, when addedto whole blood in vitro (incubated for 15 mins prior to perfusionthrough the capillary), inhibit the thrombotic process in adose-dependent manner. A concentration of 0.37 μM Compound A hasstatistically significant additional antithrombotic benefit in thepresence of aspirin and a factor Xa inhibitor, whereas thisconcentration of the P2Y₁₂ antagonist alone has no detectableantithrombotic activity in the absence of aspirin.

Example 10 Combination of Compound A and Ifetroban, a TP Antagonist, anda Factor Xa Inhibitor

Similar to the previous example, the combination of Compound A and a TPantagonist (e.g. ifetroban) and a factor Xa inhibitor showed additiveantithrombotic benefit.

In this example, blood was collected from healthy volunteers byvenipuncture into a factor Xa inhibitor C921-78 (see above). Compound Awas added to the blood sample at a concentration (1.1 μM) that mimicsthe inhibitory effects of clopidogrel in this assay. As seen in FIG. 9,when increasing concentrations of ifetroban are added to whole blood andpreincubated with Compound A (1.1 μM) for 30 mins prior to perfusion ofblood through the collagen-coated capillary (type I collagen, 1600sec⁻¹), a dose-responsive inhibition of thrombosis was observed, withthe combination of 1.1 μM of Compound A and 30 nM of ifetroban in thepresence of a factor Xa inhibitor giving the same level of inhibition asclopidogrel plus aspirin or Compound A plus aspirin.

Example 11 Combination of Compound A and a Factor Xa Inhibitor in aReal-Time Thrombosis Assay

Blood is collected by venipuncture from healthy volunteers into variousconcentrations (5-20 μM) of a factor Xa inhibitor as described herein.Increasing concentrations of Compound A are added in vitro. Following 20mins of incubation, blood is perfused through the collagen coatedcapillary (type III collagen, 1600 sec⁻¹) as described in Example 4. Itis contemplated that increasing concentrations of Compound A incombination with a fixed concentration of a selected factor Xa inhibitor(e.g., rivaroxaban, apixaban) will demonstrate inhibition of thrombosisin a dose-responsive fashion.

Example 12 Combination of Compound A and a Factor Xa Inhibitor in aThrombin Generation Assay

Whole blood from healthy volunteer donors is collected by venipunctureand drawn into 3.2% trisodium citrate (Vacutainer, Becton Dickinson) foranticoagulation. Platelet rich plasma is prepared by centrifugation. Inthe wells of a 96 well plate, the final platelet count is adjusted to150,000 μL, platelets are activated by addition of convulxin (100 ng/mL)and incubated at 37° C. for 3 mins. Subsequent to platelet activation,thrombin generation is initiated by addition of 23 pM tissue factor(Innovin, Dade Behring) and 15 mM calcium. Thrombin activity ismonitored by cleavage of the specific fluorogenic substrate(Z-gly-gly-arg-AMC, Bachem) in a fluorescence plate reader, as describedin Sinha U. et al, Inhibition of purified factor Xa amidolytic activitymay not be predictive of inhibition of in vivo thrombosis. Implicationsfor identification of therapeutically active inhibitors. ArteriosclerThromb Vasc Biol, 2003, 23:1098-1104.

It is contemplated that similar to Example 5, Compound A in combinationwith a factor Xa inhibitor (e.g., rivaroxaban, apixaban or other factorXa inhibitors) will inhibit thrombin production to a greater extent thaneither compound alone at the same concentration.

Example 13 Combination of Compound A and a Factor Xa Inhibitor InhibitsThrombosis in a Mouse Mesenteric Artery Model

Thrombosis on mouse mesenteric arteries (shear rate 1000-1300 s⁻¹) isperformed and recorded as previously described with minor modifications,as described in Andre, P. et al., Anticoagulants (thrombin inhibitors)and aspirin synergize with P2Y₁₂ receptor antagonism in thrombosis.Circulation, 2003, 108(21):2697-703. Platelets are labeled in situ usingrhodamine 6G (0.2 mg/mL) administered through the tail vein 10 minsbefore visualization of the arteries. Vessel-wall injury is triggered bya 1×1-mm filter paper saturated with a 5% FeCl₃ solution. After 5minutes, the filter paper is removed and mesenteric arteries rinsed withwarmed saline (37° C.). Platelet vessel-wall interactions are recordedfor 40 additional mins or until full occlusion occurs and persists formore than 40 seconds. C57B16J mice are orally gavaged with vehiclecontrol, Compound A (7.5, 20, 60 mg/kg) or betrixaban (4, 40, 400 mg/kg)two hours prior to vascular injury. Thrombosis is analyzed in real timeusing Simple PCI software, as described in Andre, P. et al., P2Y₁₂regulates platelet adhesion/activation, thrombus growth, and thrombusstability in injured arteries. J Clin Invest, 2003, 112(3):398-406. Thefluorescence intensity is recorded at a rate of 2 Hz for 40 minutes andplotted over time. Time to occlusion (cessation of blood flow) isanalyzed.

Doses of 20 and 60 mg/kg of Compound A prevent occlusion in response tovascular injury over the 40 min observation period. A dose of 7.5 mg/kgdelays time to occlusion of the artery. Doses contributing to plasmalevels superior to 1 μg/mL of Compound A prevent vascular occlusion,while doses achieving plasma concentration below 1 μg/mL do not preventocclusion. Doses below 200 ng/mL are non-effective doses. Doses ofbetrixaban achieving plasma concentrations superior to 1 μg/mL preventvascular occlusion. Doses below 100 ng/mL are non-effective doses. It iscontemplated that when non-effective doses of Compound A and betrixabanare combined, potent synergistic antithrombotic activities will beobtained. Similarly, other factor Xa inhibitors (e.g., rivaroxaban,apixaban) are expected to demonstrate significant antithromboticactivity in the intravital microscopy thrombosis model in mice. It iscontemplated that when non-effective doses of rivaroxaban or otherfactor Xa inhibitors as described herein are combined with non-effectivedoses of Compound A, potent synergistic antithrombotic activity will beobserved.

Example 14 Combination of Compound A and Betrixaban Inhibits Thrombosisin a Mouse Mesenteric Artery Model

Thrombosis on mouse mesenteric arteries (shear rate 1000-1300 s⁻¹) wasperformed and recorded as previously described with minor modifications.(Andre, P., et al., Anticoagulants (thrombin inhibitors) and aspirinsynergize with P2Y ₁₂ receptor antagonism in thrombosis. Circulation,2003. 108(21): p. 2697-703.) Platelets were labeled in situ usingrhodamine 6G (0.2 mg/mL) administered through the tail vein 10 minbefore visualization of the arteries. Vessel-wall injury was triggeredby a 1×1-mm filter paper saturated with a 10% FeCl₃ solution. After 5minutes, the filter paper was removed and mesenteric arteries rinsedwith warmed saline (37° C.). Platelet vessel-wall interactions wererecorded for 40 additional min or until full occlusion occurred andpersisted for more than 40 seconds. C57B16J mice were orally gavagedwith vehicle control, compound A (0.83, 2.5, 7.5, 20, 60 mg/kg) orbetrixaban (2, 4, 10 mg/kg) two hours prior to vascular injury.Thrombosis was analyzed in real time using Simple PCI software. (Andre,P., et al., P2Y ₁₂ regulates platelet adhesion/activation, thrombusgrowth, and thrombus stability in injured arteries. J Clin Invest, 2003.112(3): p. 398-406.) The fluorescence intensity was recorded at a rateof 2 Hz for 40 minutes and plotted over time. Time to occlusion(cessation of blood flow) is analyzed.

Doses of 0.83 and 2.5 mg/kg Compound A were non-effective in this model.Doses of 7.5, 20 and 60 mg/kg Compound A delayed time for appearance offirst thrombus (FIGS. 10-13) and vascular occlusion. Doses of 20 and 60mg/kg Compound A prevented occlusion in response to vascular injury overthe 40 min observation period. Doses contributing to plasma levelssuperior to 1 μg/mL Compound A prevented vascular occlusion, while dosesachieving plasma concentration below 1 μg/mL did not prevent occlusion(FIG. 13). Doses leading to plasma concentrations below 200 ng/mL werenon-effective doses in this model (FIG. 13). Doses of 2 and 4 mg/kgbetrixaban were non-effective in this model whereas doses of 10 mg/kgbetrixaban significantly delayed both time for appearance of firstthrombus and time to occlusion (FIGS. 14-17). When non-effective dosesof compound A (2.5 mg/kg) and betrixaban (2 and 4 mg/kg) were combined,potent synergistic antithrombotic activities were obtained (FIGS.14-17).

Plasma concentrations of Compound A and betrixaban were determined onblood draw collected 2 min post-occlusion or 42 min after start ofvascular injury.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

1. A method for a condition in a mammal characterized by undesiredthrombosis comprising administering to said mammal a therapeuticallyeffective amount of the following therapeutic agents: (1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; and (2) a therapeuticagent selected from the group consisting of an anticoagulant agent, anantiplatelet agent, and combinations thereof.
 2. The method of claim 1comprising administering to said mammal a therapeutically effectiveamount of the following two therapeutic agents: (1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; and (2) an anticoagulantagent.
 3. The method according to claim 2, wherein the anticoagulantagent is a factor Xa inhibitor.
 4. The method according to claim 3,wherein the factor Xa inhibitor is selected from the group consisting ofYM-150, Daiichi DU-176b,N-{(1R)-2-[4-(1-methyl-4-piperidinyl)-1-piperazinyl]-2-oxo-1-phenylethyl}-1H-indole-6-carboxamide,apixaban, rivaroxaban, otamixaban, and razaxaban.
 5. The methodaccording to claim 3, wherein the factor Xa inhibitor is selected fromthe group consisting of:


6. The method according to claim 3, wherein the factor Xa inhibitor isbetrixaban, or a pharmaceutically acceptable salt thereof.
 7. The methodaccording to claim 6, wherein the pharmaceutically acceptable salt ofbetrixaban is the maleate salt.
 8. The method according to claim 2,wherein the anticoagulant agent is selected from the group consisting ofspecific inhibitors of thrombin, factor IXa, factor XI, factor XIa orfactor VIIa.
 9. The method according to claim 2, wherein theanticoagulant agent is selected from the group consisting of AZD0837,RB2006, ximelagatran, dabigatran, bivalirudin, argatroban, lepirudin,warfarin, and phenocoumarol.
 10. The method according to claim 2,wherein the anticoagulant agent is an injectable anticoagulant agent.11. The method according to claim 2, wherein the anticoagulant agent isselected from the group consisting of synthetic pentasaccharides and lowmolecular weight heparin.
 12. The method according to claim 2, whereinthe anticoagulant agent is selected from the group consisting offondaparinux, idraparinux, biotinylated idraparinux, danaparoid,enoxaparin, dalteparin and unfractionated heparin.
 13. The methodaccording to claim 2, wherein the anticoagulant agent is an anti-factorXI antibody.
 14. The method according to claim 2, wherein theanticoagulant agent is bivalirudin.
 15. The method of claim 1 comprisingadministering to said mammal a therapeutically effective amount of thefollowing two therapeutic agents: (1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; and (2) anotherantiplatelet agent.
 16. The method according to claim 15, wherein theantiplatelet agent is an antagonist of TP receptor or a cyclooxygenaseinhibitor.
 17. The method according to claim 16, wherein thecyclooxygenase inhibitor is a reversible cyclooxygenase-1 inhibitor. 18.The method according to claim 15, wherein the antiplatelet agent isselected from the group consisting of acetylsalicylic acid, abciximab,eptifibatide, tirofiban, dipyridamole, aggrenox, cilostazol, ifetroban,isbogrel, furegrelate, resveratrol and ozagrel.
 19. The method accordingto claim 1, wherein at least one of the therapeutic agents isadministered in a sub-therapeutic dosage.
 20. The method according toclaim 1, wherein both of the therapeutic agents are administered insub-therapeutic dosages.
 21. The method according to claim 1, whereinthe two therapeutic agents are administered simultaneously.
 22. Themethod according to claim 1, wherein the two therapeutic agents areadministered sequentially.
 23. The method of claim 1, comprisingadministering to said mammal a therapeutically effective amount of thefollowing three therapeutic agents: (1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; (2) an antiplateletagent; and (3) an anticoagulant agent.
 24. The method according to claim23, wherein the antiplatelet agent is a cyclooxygenase inhibitor. 25.The method according to claim 23, wherein the antiplatelet agent isacetylsalicylic acid.
 26. The method according to claim 23, wherein theanticoagulant agent is a factor Xa inhibitor.
 27. The method accordingto claim 26, wherein the factor Xa inhibitor is betrixaban, or apharmaceutically acceptable salt thereof.
 28. The method according toclaim 27, wherein the pharmaceutically acceptable salt of betrixaban isthe maleate salt.
 29. The method according to claim 23, wherein at leastone of the therapeutic agents is administered in a sub-therapeuticdosage.
 30. The method according to claim 23, wherein all of thetherapeutic agents are administered in sub-therapeutic dosages.
 31. Themethod according to claim 23, wherein the three therapeutic agents areadministered simultaneously.
 32. The method according to claim 23,wherein the three therapeutic agents are administered sequentially. 33.The method according to claim 1, wherein the pharmaceutically acceptablesalt of[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureais the potassium salt or sodium salt.
 34. The method according to claim1, wherein said thrombosis-related condition is selected from the groupconsisting of acute myocardial infarction, unstable angina, chronicstable angina, transient ischemic attacks, strokes, peripheral vasculardisease, preeclampsia/eclampsia, deep venous thrombosis, embolism,disseminated intravascular coagulation and thrombotic cytopenic purpura,thrombotic and restenotic complications following invasive proceduresresulting from angioplasty, carotid endarterectomy, post CABG (Coronaryarteryl bypass graft) surgery, vascular graft surgery, stent placementsand insertion of endovascular devices and prosthesis.
 35. Apharmaceutical composition, comprising a pharmaceutically acceptablecarrier and the following therapeutic agents: (1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; and (2) a therapeuticagent selected from the group consisting of an anticoagulant agent, anantiplatelet agent and combinations thereof.
 36. The pharmaceuticalcomposition of claim 35, comprising a pharmaceutically acceptablecarrier and the following two therapeutic agents: (1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; and (2) an anticoagulantagent.
 37. The pharmaceutical composition according to claim 36, whereinthe anticoagulant agent is a factor Xa inhibitor.
 38. The pharmaceuticalcomposition according to claim 37, wherein the factor Xa inhibitor isselected from the group consisting of YM-150, Daiichi DU-176b,N-{(1R)-2-[4-(1-methyl-4-piperidinyl)-1-piperazinyl]-2-oxo-1-phenylethyl}-1H-indole-6-carboxamide,apixaban, rivaroxaban, otamixaban, and razaxaban or is selected from thegroup consisting of:


39. The pharmaceutical composition according to claim 37, wherein thefactor Xa inhibitor is betrixaban, or a pharmaceutically acceptable saltthereof.
 40. The pharmaceutical composition according to claim 39,wherein the pharmaceutically acceptable salt of betrixaban is themaleate salt.
 41. The pharmaceutical composition according to claim 37,wherein the anticoagulant is selected from the group consisting ofspecific inhibitors of thrombin, factor IXa, factor XI, factor XIa orfactor VIIa.
 42. The pharmaceutical composition according to claim 37,wherein the anticoagulant agent is selected from the group consisting ofAZD0837, RB2006, ximelagatran, dabigatran, bivalirudin, argatroban,lepirudin, warfarin, and phenocoumarol.
 43. The pharmaceuticalcomposition according to claim 37, wherein the anticoagulant agent is aninjectable anticoagulant agent.
 44. The pharmaceutical compositionaccording to claim 37, wherein the anticoagulant is selected from thegroup consisting of synthetic pentasaccharides and low molecular weightheparin.
 45. The pharmaceutical composition according to claim 37,wherein the anticoagulant is selected from the group consisting offondaparinux, idraparinux, biotinylated idraparinux, danaparoid,enoxaparin, dalteparin and unfractionated heparin.
 46. Thepharmaceutical composition according to claim 37, wherein theanticoagulant agent is an anti-factor XI antibody.
 47. Thepharmaceutical composition of claim 35 comprising a pharmaceuticallyacceptable carrier and the following two therapeutic agents: (1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; and (2) anotherantiplatelet agent.
 48. The pharmaceutical composition according toclaim 47, wherein the antiplatelet agent is an antagonist of TP receptoror a cyclooxygenase inhibitor.
 49. The pharmaceutical compositionaccording to claim 48, wherein the cyclooxygenase inhibitor is areversible cyclooxygenase-1 inhibitor.
 50. The pharmaceuticalcomposition according to claim 47, wherein the antiplatelet agent isselected from the group consisting of abciximab, acetylsalicylic acid,eptifibatide, tirofiban, dipyridamole, aggrenox, cilostazol, isbogrel,ifetroban, resveratrol, furegrelate and ozagrel.
 51. The pharmaceuticalcomposition according to claim 35, wherein at least one of the twotherapeutic agents is present in a sub-therapeutic dosage.
 52. Thepharmaceutical composition according to claim 35, wherein both of thetwo therapeutic agents are present in sub-therapeutic dosages.
 53. Thepharmaceutical composition of claim 35 comprising a pharmaceuticallyacceptable carrier and the following three therapeutic agents: (1)[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; (2) another antiplateletagent; and (3) an anticoagulant agent.
 54. The pharmaceuticalcomposition according to claim 53, wherein the antiplatelet agent is acyclooxygenase inhibitor.
 55. The pharmaceutical composition accordingto claim 53, wherein the antiplatelet agent is acetylsalicylic acid. 56.The pharmaceutical composition according to claim 53, wherein theanticoagulant agent is a factor Xa inhibitor.
 57. The pharmaceuticalcomposition according to claim 56, wherein the factor Xa inhibitor isbetrixaban, or a pharmaceutically acceptable salt thereof.
 58. Thepharmaceutical composition according to claim 57, wherein thepharmaceutically acceptable salt of betrixaban is the maleate salt. 59.The pharmaceutical composition according to claim 53, wherein at leastone of the therapeutic agents is administered in a sub-therapeuticdosage.
 60. The pharmaceutical composition according to claim 53,wherein all of the therapeutic agents are administered insub-therapeutic dosages.
 61. The pharmaceutical composition according toclaim 35, wherein the pharmaceutically acceptable salt of[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylureais the potassium salt or the sodium salt.
 62. A kit comprising: (1) afirst container, wherein said first container contains[4-(6-fluoro-7-methylamino-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-thiophen-2-yl-sulfonylurea,or a pharmaceutically acceptable salt thereof; and (2) a secondcontainer, wherein said second container contains an anticoagulant agentor another antiplatelet agent.
 63. The kit according to claim 62,further comprising a package insert stating that the two therapeuticagents can be used together.